Broad-spectrum affinity peptide ligand for separation and purification of influenza vaccine and application of broad-spectrum affinity peptide ligand

Abstract

The invention discloses a broad-spectrum affinity peptide ligand for separation and purification of influenza vaccines and application of the broad-spectrum affinity peptide ligand. The invention relates to a broad-spectrum affinity peptide ligand, which contains amino acid sequences which are combined with conserved structural units 130-loop, 150-loop and 190-helix at binding sites of influenza virus hemagglutinin receptors, and also contains amino acid sequences which are combined with conserved structural units 130-loop, 150-loop and 190-helix at binding sites of influenza virus hemagglutinin receptors. The amino acid sequence is R1-R2-R3-(R4) 3-R5-(R4) 2-R6-R7-R8, R1 is any one of glutamic acid, arginine, histidine and proline, R2 is tryptophan, R3 is any one of tyrosine and phenylalanine, R4 is glycine, R5 is any one of tryptophan, tyrosine and phenylalanine, and R7 is any one of tryptophan, tyrosine and phenylalanine. R6 is any one of glutamic acid, histidine, glutamine and tyrosine, R7 is any one of tryptophan and phenylalanine, and R8 is proline. The broad-spectrum affinity peptide ligand has high broad-spectrum affinity, the broad-spectrum affinity chromatography medium can obtain influenza vaccines with high purity and yield, and the broad-spectrum affinity chromatography medium has great potential to be applied to the separation and purification process of the influenza vaccines.

Description

Technical Field

[0001] The present invention relates to the field of vaccine separation and purification, and in particular to a broad-spectrum affinity peptide ligand for separation and purification of influenza vaccines and application thereof. Background Art

[0002] Vaccination is still the best strategy for preventing influenza in the elderly and children. The current influenza vaccine preparation systems mainly include chicken embryo cells and mammalian cells. In the preparation of influenza vaccines, ultracentrifugation, gel filtration chromatography, and ion exchange chromatography are still the most commonly used unit operations for separation and purification. They are not only laborious, time-consuming, and energy-intensive, but also have poor selectivity and limited processing capacity. The ultracentrifugation process is very difficult to scale up. The development of broad-spectrum affinity chromatography technology is undoubtedly an important method to overcome the above problems.

[0003] Affinity chromatography technology has been widely used in antibody purification processes, but there are few successful cases in the purification of virus-like particles, including vaccines and lentiviruses. This is mainly due to the large particle size, complex structure and large number of mutants of virus particles, which brings great challenges to the development of affinity chromatography ligands.

[0004] Hemagglutinin (HA) is an important and abundant glycoprotein on the surface of influenza virus, and therefore is an important target for designing small molecule drugs and vaccine purification affinity ligands. Liang Guizhao et al. successively disclosed patents (CN201010540985.9 and CN201010541077.1) for obtaining hemagglutinin protein affinity peptides of two influenza A type H1N1 viruses and influenza A type H3N2 viruses using phage screening technology. Teruhiko et al. reported the use of phage display technology combined with molecular docking to obtain the peptide LVRPLAL as an anti-influenza virus peptide ligand (Bioorganic & Medicinal Chemistry, 2016, 24 (5): 1106-14.). However, the above affinity peptide molecules and peptide ligands do not have broad spectrum, and the high variability of HA antigen is an important reason why influenza problems have long troubled everyone. Due to the lack of a broad-spectrum affinity ligand that can be applied to different serotypes of influenza vaccines, the application of affinity chromatography technology in the existing influenza vaccine preparation process has been greatly limited. Therefore, developing a new type of broad-spectrum affinity ligand for influenza vaccine and synthesizing a broad-spectrum affinity chromatography medium based on it has become a top priority in the influenza vaccine preparation process. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is that there is a lack of broad-spectrum affinity ligands with excellent performance in the current influenza vaccine purification process, so the needs of producing influenza virus vaccines of different serotypes cannot be effectively met; in view of the above problems, the present invention provides a broad-spectrum affinity peptide ligand for separation and purification of influenza vaccines, and a broad-spectrum affinity chromatography medium synthesized with the broad-spectrum affinity ligand and its application, and provides a preparation method of the broad-spectrum affinity chromatography medium.

[0006] A broad-spectrum affinity peptide ligand contains amino acid sequences that bind to the conservative structural units 130-loop, 150-loop and 190-helix of the influenza virus hemagglutinin receptor binding site.

[0007] The amino acid sequence is R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 ;

[0008] Among them, R 1 is any one of glutamic acid, arginine, histidine and proline, R 2 Tryptophan, R 3 is either tyrosine or phenylalanine, R 4 is glycine, R 5 is any one of tryptophan, tyrosine and phenylalanine, R 6 is any one of glutamic acid, histidine, glutamine and tyrosine, R 7 is either tryptophan or phenylalanine, R 8 For proline.

[0009] As a parallel embodiment, the amino acid sequence of the broad-spectrum affinity peptide ligand is: PWFGGGYGGEWP, HWFGGGYGGQWP or RWYGGGWGGHFP.

[0010] A broad-spectrum affinity chromatography medium comprises a solid phase matrix and the broad-spectrum affinity peptide ligand coupled to the solid phase matrix.

[0011] The solid phase matrix is ​​a thiopyridinium-treated solid phase matrix.

[0012] A method for preparing a broad-spectrum affinity chromatography medium, comprising:

[0013] Access to broad-spectrum affinity peptide ligands and solid-phase matrices;

[0014] A solution containing a broad-spectrum affinity peptide ligand is mixed with a solid phase matrix to obtain a broad-spectrum affinity chromatography medium through a coupling reaction.

[0015] The coupling reaction process is as follows: an amino acid group for coupling is introduced into the C-terminus of the broad-spectrum affinity peptide ligand, and a solution containing the broad-spectrum affinity peptide ligand is prepared by using a cross-linking buffer; a solid phase matrix is ​​thiopyridinated, and the thiopyridinated solid phase matrix and the solution containing the broad-spectrum affinity peptide ligand are mixed and reacted in a water bath shaker.

[0016] The amino acid groups used for coupling are three glycines and one cysteine ​​which are sequentially connected to the C-terminus of the broad-spectrum affinity peptide ligand;

[0017] And / or, the process of mercapto pyridylating the solid phase matrix is:

[0018] (1) Activation: The solid phase matrix is ​​washed and activated with deionized water and an activating substance in sequence, and an activated solid phase matrix is ​​obtained after the reaction.

[0019] (2) Thiolation: The activated solid phase matrix is ​​reacted with a phosphate buffer and a sodium thiosulfate solution for the first reaction, and then a sodium bicarbonate solution and an ethylenediaminetetraacetic acid solution containing dithiothreitol are sequentially added for the second reaction, and then washed with sodium bicarbonate and ethylenediaminetetraacetic acid solutions in turn to obtain a thiolated solid phase matrix;

[0020] (3) Pyridylation: The thiolated solid phase matrix is ​​cleaned, and then an acetone cleaning solution and a dithiodipyridyl solution are added in sequence, mixed and reacted, and finally cleaned and dried to obtain a thiopyridyl solid phase matrix.

[0021] In the activation step, the activation substances are dimethyl sulfoxide, epichlorohydrin and sodium hydroxide solution, wherein the amount of dimethyl sulfoxide added is 0.4-2 times the mass of the solid matrix, the amount of epichlorohydrin added is 0.2-1 times the mass of the solid matrix, the amount of sodium hydroxide solution added is 0.2-1 times the mass of the solid matrix, the volume concentration of dimethyl sulfoxide is 20%-100%, and the mass concentration of the sodium hydroxide solution is 0.5-1 mol / L; the activation reaction time is 3-6h.

[0022] In the thiolation step: the concentration of the phosphate buffer is 0.1-0.5 mol / L, and the pH value is 6-7; the concentration of the sodium thiosulfate solution is 0.5-2 mol / L; the amount of the phosphate buffer and / or the sodium thiosulfate solution added is 0.4-2 times the mass of the activated solid phase matrix; the time of the first reaction is 3-24 hours; the concentration of the sodium bicarbonate solution is 0.1-0.2 mol / L, the concentration of the ethylenediaminetetraacetic acid solution is 0.5-1 mmol / L, and the amount of dithiothreitol added to the ethylenediaminetetraacetic acid solution is 0.5-1 g / L; the amount of the two solutions added is 0.5-1 times the mass of the activated solid phase matrix; the time of the second reaction is 0.5-1.5 hours;

[0023] In the pyridation step, the acetone cleaning solution is an acetone solution with a volume concentration of 50%-70%, and the acetone cleaning solution contains 0.5-1mmol / L ethylenediaminetetraacetic acid and 10-50mmol / L sodium bicarbonate; the mass concentration of the disulfide pyridine solution is 0.1-0.5mol / L; the amount of the acetone cleaning solution added is 1-2 times the mass of the thiolated solid phase matrix, and the amount of the disulfide pyridine solution added is 2-4 times the mass of the thiolated solid phase matrix; the reaction time is 1-3h; in the final cleaning step, the acetone solution with a volume concentration of 50%-70% and the 0.5-1mmol / L ethylenediaminetetraacetic acid solution are used for cleaning.

[0024] Application of the above broad-spectrum affinity chromatography medium in the separation and purification of influenza vaccines.

[0025] The separation and purification process is as follows: the biological raw material liquid containing influenza vaccine components flows into the above-mentioned broad-spectrum affinity chromatography medium, and after adsorption, washing and elution steps, the eluted components are collected to obtain the influenza vaccine.

[0026] The adsorption buffer used in the adsorption process is a 0.01-0.05 mol / L PB buffer with a pH of 5.0-6.5 and containing 0-0.3 mol / L NaCl; the low pH elution buffer used in the elution process is a 0.01-0.10 mol / L Gly-HCl buffer with a pH of 2.5-4.0.

[0027] The technical solution of the present invention has the following advantages:

[0028] 1. The broad-spectrum affinity peptide ligand provided by the present invention has a high affinity to the influenza virus hemagglutinin protein receptor binding site and binds to the conservative structural units 130-loop, 150-loop and 190-helix of the influenza virus hemagglutinin receptor binding site; based on the influenza virus hemagglutinin protein receptor binding site, a polypeptide library is constructed by a de novo design strategy, and a broad-spectrum affinity peptide ligand capable of binding to the conservative structural units 130-loop, 150-loop and 190-helix of the influenza virus hemagglutinin receptor binding site is obtained through four rounds of molecular simulation screening; the broad-spectrum affinity peptide ligand can specifically bind to the hemagglutinin protein and the inactivated influenza vaccine, effectively overcoming the problem that the existing chromatography ligand is not applicable to HA antigens with high variability, thereby resulting in poor broad-spectrum affinity.

[0029] 2. The present invention provides a broad-spectrum affinity peptide ligand that can effectively bind to the conservative structural units 130-loop, 150-loop and 190-helix of the influenza virus hemagglutinin receptor binding site, and the amino acid sequence structure of the broad-spectrum affinity peptide ligand is R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 ; It has been verified that the broad-spectrum affinity peptide ligand can specifically bind to hemagglutinin protein and inactivated influenza vaccine and has broad-spectrum affinity; using it as a broad-spectrum affinity ligand to synthesize a broad-spectrum affinity chromatography medium, influenza vaccine can be harvested with higher purity and yield, and the broad-spectrum affinity chromatography medium has great potential for application in the separation and purification process of influenza vaccine.

[0030] 3. The broad-spectrum affinity chromatography medium provided by the present invention couples a broad-spectrum affinity peptide ligand having a high affinity for the influenza virus hemagglutinin protein receptor binding site with a thiopyridinium-modified solid phase matrix, such as an agarose gel medium, to prepare a broad-spectrum affinity chromatography medium for separation and purification of influenza vaccines; the broad-spectrum affinity chromatography medium has the advantages of good versatility, good specificity, simple medium preparation and low process cost.

[0031] 4. The present invention provides the use of the broad-spectrum affinity chromatography medium of the present invention in the separation and purification of influenza vaccines, which can efficiently separate and purify influenza vaccine components from a complex raw material liquid, with almost no adsorption of individual impurities. At a relatively high yield, the purity of the separated vaccine can reach more than 92%, or even more than 95%, and the separation effect is very significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 is a diagram of the process of obtaining the broad-spectrum affinity peptide ligand in Example 1 of the present invention;

[0034] Figure 2 is a graph showing the amino acid mapping results of different docking regions in step (1) of Example 1 of the present invention; wherein a is the amino acid mapping result of the 130-loop region, b is the amino acid mapping result of the 150-loop region, and c is the amino acid mapping result of the 190-helix region;

[0035] Figure 3 is a docking conformation diagram of different amino acid residues in step (1) of Example 1 of the present invention; wherein d is the docking conformation of E, W, and P in the 130-loop region, e is the docking conformation of Y in the 150-loop region, and f is the docking conformation of P, W, and F in the 190-helix region;

[0036] Figure 4 is a molecular docking score distribution diagram of the polypeptide library in step (2) of Example 1 of the present invention; wherein a is the docking score distribution of Autodock vina, b is the docking score distribution of HADDOCK, and c is the docking score distribution of ROSETTAFlexPepDock;

[0037] Figure 5 is a graph of molecular dynamics simulation and MM / PBSA calculation results in step (3) of Example 1 of the present invention;

[0038] Figure 6 The figure is a micro-thermophoresis result diagram of the top three polypeptides ranked by MM / PBSA and the hemagglutinin protein in step (4) of Example 1 of the present invention;

[0039] Figure 7 It is a chromatography result diagram of separating hemagglutinin protein from ovalbumin by PWFSep-6FF in Example 2 of the present invention and an SDS-PAGE analysis diagram of the affinity chromatography elution result;

[0040] Figure 8 It is a graph showing the chromatography results of separating inactivated influenza A vaccine from Vero cell supernatant by PWFSep-4FF in Example 2 of the present invention and an SDS-PAGE analysis graph showing the affinity chromatography elution results. DETAILED DESCRIPTION

[0041] Example 1

[0042] A broad-spectrum affinity peptide ligand, the process of obtaining it is as follows Figure 1 As shown, the details are as follows:

[0043] (1) Construction of peptide library

[0044] The receptor binding site of the hemagglutinin protein was used as a template, and the amino acid positioning method was used to generate short peptide fragments. The 19 amino acids except Cys were docked to the conservative structural units 130-loop, 150-loop and 190-helix of the hemagglutinin receptor binding site respectively; the binding free energy of the amino acid and the corresponding region was used as the standard for screening. Figure 2 Where ac is the binding free energy of the amino acid in the three docking regions, and those below -2.5 kcal / mol are listed as candidate amino acids.

[0045] Among them, Figure 2 As shown in a, in addition to W, E, H, Q, R, Y, F, P, and L also meet the screening criteria. Then, according to the binding positions of the above 9 candidate amino acids in the 130-loop region, they are divided into three groups: EHQYR, LWF, and P. Thus, 15 combinations of tripeptide fragments can be obtained; Figure 3 As shown in d, the binding positions of three groups of amino acids in the 130-loop region are shown, represented by the three amino acids E, W, and P. The same method, such as Figure 2 As shown in b, the amino acids that meet the criteria in the 150-loop region are F, W, and Y; Figure 3 As shown in e, the three amino acids are bound between Trp152 and Leu153. Figure 2 As shown in c, the amino acids that meet the criteria in the 190-helix region are D, E, H, R, N, W, F, P, I, and Y, which are divided into three groups, DEHNIPR, W, and FY, according to the binding position, thus 14 tripeptide fragment combinations can be obtained; Figure 3 f shows the binding positions of three groups of amino acids in the 190-helix region, represented by the three amino acids P, W, and F.

[0046] Glycine is used as a linker between short peptide fragments. If the distance between the C / N terminus of one amino acid residue and the N / C terminus of another residue is less than (i.e. the sum of the lengths of a peptide bond and two amino acid backbones), then an amino acid residue can be inserted between these two residues; if the distance between them is and If the distance is greater than Then 3 or more residues can be inserted. The maximum distance between the amino acids bound in the 130-loop region and the amino acids bound in the 150-loop region is Therefore, two glycines can be inserted, and the maximum distance between the 150-loop amino acid and the 190-helix amino acid is Therefore, three glycines can be inserted. So far, the basic model of the peptide has been determined. According to statistics, there are 630 peptide ligands in the peptide library for the next screening process.

[0047] (2) Molecular docking screening of peptide libraries

[0048] The first round of screening was performed using Autodock vina for semi-flexible docking, such as Figure 4 As shown in a, the binding free energy between peptide ligand and hemagglutinin protein is in the range of -5.8 to -11.3 kcal / mol. Peptide ligands with binding free energy lower than -8.7 kcal / mol were selected for the next round of screening, and 314 peptides were screened so far. In order to further explore the affinity of peptide ligand and design site, the second round of screening used HADDOCK for local semi-flexible docking, as shown in Figure 4 As shown in Figure b, the binding free energy of the peptide and RBS is between -71.8 and -110.1 kJ / mol. For the convenience of screening, the influenza A virus broad-spectrum antibody C05 was used as a positive control, and its HADDOCK score was -91.8 kJ / mol. Those with scores below this score were listed as candidate peptide ligands. So far, 114 peptide ligands have been screened. The structure of the peptide ligand was determined to be R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 ; Among them, R 1 is any one of glutamic acid, arginine, histidine and proline, R 2 Tryptophan, R 3 is either tyrosine or phenylalanine, R 4 is glycine, R 5 is any one of tryptophan, tyrosine and phenylalanine, R 6 is any one of glutamic acid, histidine, glutamine and tyrosine, R 7 is either tryptophan or phenylalanine, R 8 For proline.

[0049] Both vina and HADDOCK are semi-flexible molecular dockings and cannot take into account the impact of protein side chain flexibility. The ROSETTA FlexPepDock docking program can take into account the flexibility of polypeptides and the side chain flexibility of receptor proteins, and is usually used to optimize the binding conformation between peptide ligands and target proteins. If the complex result of the target protein and its ligand is known, then ROSETTA FlexPepDock can achieve high-precision simulation of the complex. In the present invention, the interface energy score (I_sc) is used to evaluate the affinity between the 114 peptide ligands obtained in the previous round and HA. The I_sc item includes hydrogen bonds, Lennard–Jones attraction and repulsion energy, lazaridis-jarplus solvation energy, and salt bridge effects. In order to improve the accuracy of docking, each peptide ligand and protein were docked three times in parallel, and the docking results are as follows. Figure 4 c, and finally the top ten peptides were obtained for the next step of molecular dynamics simulation screening. The sequences of the top ten peptides are shown in SEQ No.1-10, and the three parallel docking results of the top ten peptides are shown in Table 1.

[0050] Table 1

[0051]

[0052] (3) Molecular dynamics simulation and MM / PBSA free energy calculation

[0053] In order to further detect the affinity of peptides and hemagglutinin proteins and the microscopic molecular behavior of their peptide-hemagglutinin complexes in solution, 10 peptide ligand-hemagglutinin complexes obtained by molecular docking screening were subjected to 100ns molecular dynamics simulations. The conformations of the 10 peptide and hemagglutinin protein complexes obtained by ROSETTA FlexPepDock were used as the initial conformations. The simulation was performed using the software GROMACS2022.2 under constant temperature, constant pressure and periodic boundary conditions. The Amber99sb-ildn all-atom force field was used to place the complex in a 12.3nm×4.75nm×3.8nm box. The TIP3P water model was used, and then the Na required to balance the net charge of the system and stabilize the buffer solution (150mM NaCl) was added. + and Cl -; Then, energy minimization was performed to remove the collisions between atoms and incorrect geometric configurations in the system; then, restricted dynamic equilibrium was performed in the canonical (NVT) ensemble for 1000ps and the isothermal and isobaric (NPT) ensemble for 1000ps, and finally, an unrestricted MD simulation was performed for 100ns. All hydrogen bonds involved were constrained using the LINCS algorithm with an integration step of 2fs. The electrostatic interaction was calculated using the Particle-mesh Ewald (PME) method with a cutoff of 1.2nm and a non-bonded interaction cutoff of 0. The simulation temperature was controlled to 295 K using the V-rescale temperature coupling method, and the pressure was controlled to 1 bar using the Parrinello-Rahman barostat method. After the simulation, the last 20 ns were selected to extract the conformation, and the free energy calculation was performed using the MM / PBSA method.

[0054] First, the minimum distance between the peptide and the hemagglutinin protein and the change in the average number of atomic contacts during the simulation were calculated. The minimum distance calculation results are shown in Figure 5 a, the calculated results of the average atomic contact number are as follows Figure 5 As shown in b. The results showed that, except for HWFGGGWGGRWP, the minimum distance between the other nine polypeptides and the hemagglutinin protein was between 0.17 and 0.28 nm, and the number of contacts was stable at about 2000. In addition, Figure 5 The MM / PBSA results shown in c show that, except for HWFGGGWGGRWP, the binding free energies of the other nine peptide ligands and the hemagglutinin protein are all negative, indicating that the peptide ligands and the hemagglutinin protein are tightly bound during the entire molecular dynamics simulation process, proving that these nine peptide ligands may be effective broad-spectrum affinity peptide ligands.

[0055] (4) Microthermophoresis affinity characterization

[0056] The top three broad-spectrum affinity peptide ligands PWFGGGYGGEWP, HWFGGGYGGQWP and RWYGGGWGGHFP ranked in MM / PBSA were selected for affinity characterization.

[0057] Specifically, the Monolith NT.115 microthermophoresis instrument of Nano Temper was used to determine the binding constant between the peptide and the protein. His-HA was labeled using the Nano Temper His-Tag labeling kit RED-tris-NTA. 100 μL HA protein (200 nmol / L) and 100 μL dye (100 nmol / L) were mixed and incubated at room temperature for 30 minutes. The sample was centrifuged at 4°C and 15000g for ten minutes, and the supernatant was taken to a new tube. In this experiment, the final experimental concentration of HA was 50 nmol / L, and the broad-spectrum affinity peptide ligand was diluted from 100 μmon / L to obtain 16 different concentrations. The HA protein and the broad-spectrum affinity peptide ligand solution were mixed in a volume ratio of 1:1 to obtain a 20 μL mixture, and the sample was aspirated with a capillary tube for detection. The signal was measured at medium power and 40% LED power. The change of fluorescence with concentration was detected from high concentration to low concentration, and a series of concentration-fluorescence curves were obtained. The dissociation constant K of the broad-spectrum affinity peptide ligand and HA protein was obtained by fitting using MO.Control and MO.Affinity Analysis software. d .like Figure 6 As shown, the affinity of PWFGGGYGGEWP to three different influenza A virus HA proteins is stable at 0.58-0.73 μmol / L, HWFGGGYGGQWP is stable at 0.94-2.01 μmol / L, and RWYGGGWYYHFP is stable at 2.19-7.27 μmol / L; this is sufficient to prove that the broad-spectrum affinity peptide ligands screened by the present invention have better affinity for different influenza A virus hemagglutinin proteins.

[0058] Example 2

[0059] A broad-spectrum affinity chromatography medium containing a broad-spectrum affinity peptide ligand, PWFGGGYGGEWP with the highest affinity is selected for subsequent chromatography separation and purification experiments. The acquisition process and experimental process of the broad-spectrum affinity chromatography medium are as follows:

[0060] (1) Obtaining a thiopyridinium-containing solid phase matrix

[0061] Sepharose stored in 20% ethanol TM6Fast Flow (IMAC chromatography filler) was rinsed with deionized water and DMSO and drained. 3g of medium was weighed and 6mL of dimethyl sulfoxide (DMSO), 3mL of epichlorohydrin (ECH) and 6mL of 1mol / L NaOH were added in sequence. The mixture was incubated in a water bath shaker at 25°C and 170rpm for 3h. Then, it was rinsed with deionized water and drained. 6mL of 0.5mol / L PB (pH 6.3) and 3mL of 2mol / L Na2S2O3 were added in sequence. The mixture was incubated for 6h under the same conditions. After the reaction was completed, it was rinsed with deionized water and drained. 3mL of 0.2mol / L NaHCO3 and 3mL of 1mmol / L EDTA containing 0.5g of dithiothreitol (DTT) were added in sequence. The mixture was incubated in a water bath shaker for 30min under the same conditions. After the reaction is completed, 0.2mol / L NaHCO3, 1mmol / L EDTA and acetone cleaning solution (60% acetone solution containing 1mmol / L EDTA and 20mmol / L NaHCO3) are used to wash in sequence, and then 5mL of acetone cleaning solution and 10mL of 0.3mol / L 2,2'-dithiodipyridine solution are added in sequence and incubated for 1h. After the reaction is completed, 60% acetone solution, 1mmol / EDTA and 0.1mol / L Tris-HCl are used to wash the medium in sequence to obtain a thiopyridyl solid phase matrix.

[0062] (2) Obtaining broad-spectrum affinity chromatography media

[0063] Three glycines and one cysteine ​​are introduced into the C-terminus of the broad-spectrum affinity peptide ligand as coupling amino acid groups, wherein the method of introducing three glycines and one cysteine ​​into the C-terminus of the polypeptide is a routine method for those skilled in the art and will not be described in detail here.

[0064] Using 0.1 mol / L Tris-HCl as the cross-linking buffer, 20 mg of the above broad-spectrum affinity peptide ligand containing coupled amino acid groups and 0.6 g of thiopyridinium-modified solid phase matrix were reacted in a water bath shaker for 1.5 h. After the reaction, an appropriate amount of cysteine ​​was added and incubated for 30 min under the same conditions to block the unreacted active groups of PWFGGGYGGEWP. After the reaction, the medium was named PWFSep-6FF.

[0065] (3) Obtaining broad-spectrum affinity chromatography media for virus isolation and purification experiments

[0066] Sepharose is used as the solid matrix for virus separation and purification experiments. TM 4Fast Flow, except that the remaining steps are the same as the above steps (1) and (2), and the final synthesized broad-spectrum affinity chromatography medium is named PWFSep-4FF.

[0067] (4) Affinity chromatography experiment

[0068] HA and inactivated influenza vaccine were separated and purified from ovalbumin (OVA) and Vero cell supernatant on a Tricorn 5 / 50 column containing 1 mL of broad-spectrum affinity chromatography medium. The adsorption buffer was 20 mmol / L PBS buffer (100 mmol / L NaCl, pH 6.0). HA and OVA were dissolved in the adsorption buffer and incubated for 2 h, with the final concentrations of HA and OVA being 1 mg / mL and 2 mg / mL, respectively. Inactivated influenza vaccine and Vero cell supernatant were dissolved in the adsorption buffer and incubated for 2 h, with the final concentrations of inactivated influenza vaccine and Vero cell supernatant being 1 mg / mL and 2 mg / mL, respectively.

[0069] Specifically, after filling a Tricorn 5 / 5 column with PWFSep-6FF medium and equilibrating with adsorption buffer for at least 10 column volumes, the sample (OVA+HA) was loaded at a flow rate of 0.5 mL / min and eluted with 0.1 mol / L Gly-HCl (pH 3.0) at a flow rate of 0.8 mL / min, and the absorbance at 280 nm was measured using a UV-900 detector. PWFSep-6FF had almost no adsorption of individual impurities, but an elution peak was generated after the mixture was passed through. The hemagglutinin protein was successfully separated and purified by SDS-PAGE analysis of the eluted fractions, as shown in the following results. Figure 7 As shown, a is the chromatography result diagram, and b is the electrophoresis result diagram;

[0070] Figure 7 In a, W stands for outflow, E stands for elution, and R stands for regeneration; Figure 7 In b, M represents protein marker, F represents feedstock, W represents washing fraction, and E represents eluted fraction; by measuring protein concentration and grayscale analysis, the recovery rate of hemagglutinin protein is 88.3%-92.2%, and the purity is 92.3%-95.7%.

[0071] At the same time, the inactivated influenza vaccine purification experiment was conducted using the sample (Vero cell supernatant + inactivated influenza vaccine). Except that the medium was PWFSep-4FF, the adsorption buffer was changed to 20mmol / L PBS buffer (300mmol / L NaCl, pH6.0). Other than that, all other steps were the same as above. The results of the inactivated influenza vaccine purification experiment showed that under the new medium and new adsorption conditions, PWFSep-4FF had almost no adsorption of individual impurities, but an elution peak was generated after the mixture was passed through. The inactivated influenza vaccine was successfully separated and purified by SDS-PAGE analysis of the elution components. The results are shown in Figure 2. Figure 8 As shown, a is the chromatography result diagram, and b is the electrophoresis result diagram; Figure 8 In b, M represents protein marker, F represents feedstock, FT represents washing fraction, and E represents eluted fraction; by measuring protein concentration and grayscale analysis, the recovery rate of hemagglutinin protein is 68.3%-72.2%, and the purity is 95.9%-97.2%.

[0072] Therefore, the above experiments further show that the peptide ligand PWFGGGYGGEWP can effectively purify influenza A vaccine, and it is a broad-spectrum affinity peptide ligand with high affinity. Affinity chromatography experiments prove that the broad-spectrum affinity chromatography medium coupled with the PWFGGGYGGEWP broad-spectrum affinity peptide ligand in the present invention can obtain influenza vaccine with high purity and yield, thereby proving that the broad-spectrum affinity chromatography medium has great potential for application in influenza vaccine separation and purification processes.

[0073] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A broad-spectrum affinity peptide ligand, characterized in that: It contains the amino acid sequence of the conserved structural units 130-loop, 150-loop and 190-helix that bind to the influenza virus hemagglutinin receptor binding site.

2. The broad-spectrum affinity peptide ligand according to claim 1, characterized in that The amino acid sequence is R 1 -R 2 -R 3 -(R 4 )3-R 5 -(R 4 )2-R 6 -R 7 -R 8 ; Among them, R 1 is any one of glutamic acid, arginine, histidine and proline, R 2 Tryptophan, R 3 is either tyrosine or phenylalanine, R 4 is glycine, R 5 is any one of tryptophan, tyrosine and phenylalanine, R 6 is any one of glutamic acid, histidine, glutamine and tyrosine, R 7 is either tryptophan or phenylalanine, R 8 For proline.

3. The broad-spectrum affinity peptide ligand according to claim 2, characterized in that The amino acid sequence of the broad-spectrum affinity peptide ligand is: PWFGGGYGGEWP, HWFGGGYGGQWP or RWYGGGWGGHFP.

4. A broad-spectrum affinity chromatography medium, characterized in that: The invention comprises a solid phase matrix and a broad-spectrum affinity peptide ligand as claimed in any one of claims 1 to 3 coupled to the solid phase matrix.

5. The broad-spectrum affinity chromatography medium according to claim 4, characterized in that The solid phase matrix is ​​a thiopyridinium-treated solid phase matrix.

6. The method for preparing the broad-spectrum affinity chromatography medium according to claim 4 or 5, characterized in that: include: Access to broad-spectrum affinity peptide ligands and solid-phase matrices; A solution containing a broad-spectrum affinity peptide ligand is mixed with a solid phase matrix to obtain a broad-spectrum affinity chromatography medium through a coupling reaction.

7. The preparation method according to claim 6, characterized in that: The coupling reaction process is as follows: an amino acid group for coupling is introduced into the C-terminus of the broad-spectrum affinity peptide ligand, and a solution containing the broad-spectrum affinity peptide ligand is prepared by using a cross-linking buffer; a solid phase matrix is ​​thiopyridinated, and the thiopyridinated solid phase matrix and the solution containing the broad-spectrum affinity peptide ligand are mixed and reacted in a water bath shaker.

8. The preparation method according to claim 7, characterized in that: The amino acid groups used for coupling are three glycines and one cysteine ​​which are sequentially connected to the C-terminus of the broad-spectrum affinity peptide ligand; And / or, the process of mercapto pyridinylation of the solid phase matrix is: (1) Activation: The solid phase matrix is ​​washed and activated with deionized water and an activating substance in sequence, and an activated solid phase matrix is ​​obtained after the reaction; (2) Thiolation: The activated solid phase matrix is ​​reacted with a phosphate buffer and a sodium thiosulfate solution for the first reaction, and then a sodium bicarbonate solution and an ethylenediaminetetraacetic acid solution containing dithiothreitol are sequentially added for the second reaction, and then washed with sodium bicarbonate and ethylenediaminetetraacetic acid solutions in turn to obtain a thiolated solid phase matrix; (3) Pyridylation: The thiolated solid phase matrix is ​​cleaned, and then an acetone cleaning solution and a dithiodipyridyl solution are added in sequence, mixed and reacted, and finally cleaned and dried to obtain a thiopyridyl solid phase matrix.

9. The preparation method according to claim 8, characterized in that: In the activation step, the activation substances are dimethyl sulfoxide, epichlorohydrin and sodium hydroxide solution, wherein the amount of dimethyl sulfoxide added is 0.4-2 times the mass of the solid matrix, the amount of epichlorohydrin added is 0.2-1 times the mass of the solid matrix, the amount of sodium hydroxide solution added is 0.2-1 times the mass of the solid matrix, the volume concentration of dimethyl sulfoxide is 20%-100%, and the mass concentration of the sodium hydroxide solution is 0.5-1 mol / L; the activation reaction time is 3-6h; In the thiolation step: the concentration of the phosphate buffer is 0.1-0.5 mol / L, and the pH value is 6-7; the concentration of the sodium thiosulfate solution is 0.5-2 mol / L; the amount of the phosphate buffer and / or the sodium thiosulfate solution added is 0.4-2 times the mass of the activated solid phase matrix; the time of the first reaction is 3-24 hours; the concentration of the sodium bicarbonate solution is 0.1-0.2 mol / L, the concentration of the ethylenediaminetetraacetic acid solution is 0.5-1 mmol / L, and the amount of dithiothreitol added to the ethylenediaminetetraacetic acid solution is 0.5-1 g / L; the amount of the two solutions added is 0.5-1 times the mass of the activated solid phase matrix; the time of the second reaction is 0.51.5 hours; In the pyridation step, the acetone cleaning solution is an acetone solution with a volume concentration of 50%-70%, and the acetone cleaning solution contains 0.5-1mmol / L ethylenediaminetetraacetic acid and 10-50mmol / L sodium bicarbonate; the mass concentration of the disulfide pyridine solution is 0.1-0.5mol / L; the amount of the acetone cleaning solution added is 1-2 times the mass of the thiolated solid phase matrix, and the amount of the disulfide pyridine solution added is 2-4 times the mass of the thiolated solid phase matrix; the reaction time is 13h; in the final cleaning step, the acetone solution with a volume concentration of 50%-70% and the ethylenediaminetetraacetic acid solution with a volume concentration of 0.5-1mmol / L are used for cleaning.

10. Use of the broad-spectrum affinity chromatography medium according to claim 4 or 5 in separation and purification of influenza vaccines.

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